Catalytic RNAs, like the group IIB intron ribozyme of S. cerevesiae, require a high magnesium(II) concentration to show folding and function in vitro . In contrast, in vivo conditions are characterized by a highly crowded cellular environment and much lower ion concentration. Molecular crowding agents are a widespread tool to mimic cellular crowding . However, particular physical/chemical properties explaining the crowders influence are mostly not understood. In this study, we gain new insights on how polymer properties like viscosity, pore size etc. influence the activity and folding of a large RNA.
We combined bulk activity assays and single-molecule Förster Resonance Energy Transfer experiments, screening the PEG volume fraction (%) and molecular weight (MW). Our results revealed that upon the influence of crowding agents, a compaction of the underlying structure depends on the PEG % and the presence of different PEG MW and % unveiled an optimal pore size in terms of catalytic activity. In summary, an increasing density of the crowding environment shifts the RNA towards the most compact state, but the ribozyme is only active if the crowders network matches its size . We interpret the most compact state as necessary, but not sufficient, to keep the ribozyme active.
Financial support from the European Research Council (MIRNA N° 259092, to RKOS), the Swiss National Fund (SNF), and the Forschungskredit Grant of the University of Zürich (FK-14-096 and 15-092 to RB) are gratefully acknowledged.
 Swisher J.F., Su L.J., Brenowitz M., Anderson V.E., Pyle A.M., J. Mol. Bio., 315, 297-310 (2002).
 Kilburn D., Roh J.H., Guo L., Briber R.M., Woodson S.A., JACS, 132, 8690-6 (2010).
 Steiner M., Karunatilaka K.S., Sigel R.K.O., Rueda D., Proc. Natl. Acad. Sci. U.S.A.,105, 13853-8 (2008).
 aBörner R, Fiorini E, Sigel R.K.O., Chimia, 69, 207-212 (2015).; bFiorini E., Paudel B., Börner R., Rueda D., Sigel R.K.O., submitted.
 König S.L.B., Hadzic M., Fiorini E., Börner R., Kowerko D., Blanckenhorn W.U., Sigel R.K.O., PLoS ONE, 8, e84157 (2013).
The popularity of carbocyanine dyes in single molecule spectroscopy of nucleic acids is unbroken . Studying the dynamics of large RNA constructs and the binding kinetics such as the exon/intron binding side interaction of the group II intron in S. Cerevisiae [2,3] have motivated a thorough photophysical characterization of the FRET pair Cy3/Cy5 in context of nucleic acids and RNA in particular. We show that Mg2+ as a mediator of RNA-dye interactions enhances the cyanine fluorescence lifetime. The increasing window for depolarization as monitored by time-resolved anisotropy further revealed a dynamic equilibrium between free tumbling and stacking on the RNA backbone, with the stacked conformation preventing photoisomerization . Tracking fluorophore mobility covalently bound to the RNA on an atomistic level by means of molecular dynamics  allow to disentangle different types of dye-dye and dye-RNA interactions. Our hybrid approach combining time-correlated single photon counting and computer simulations will benefit the interpretation of absolute distance measurement by smFRET.
 M.Levitus and S.Ranjit, Q. Rev. Biophys 2011, 44, 123-151.
 D.Kowerko, S.L.B.König, M.Skilandat, D.Kruschel, M.C.A.S.Hadzic, L.Cardo, R.K.O.Sigel, PNAS 2015, 112, 3403-3408.
 M. Khier, D. Kowerko, F. Steffen, R. Börner and R.K.O.Sigel, in preparation.
 F.Steffen, R.K.O. Sigel, R.Börner, in preparation.
 R.Best, H. Hofmann, D. Nettels, B. Schuler, Biophys J 2015, 11,2721-2731.
Proc. SPIE. 9711, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IX
KEYWORDS: Point spread functions, Detection and tracking algorithms, Data modeling, Imaging systems, Cameras, Image processing, Luminescence, Molecules, Fluorescence spectroscopy, Signal processing, Solids, Video processing, Charge-coupled devices, Fluorescence resonance energy transfer, Signal detection, Molecular energy transfer, Data analysis
The processing and analysis of surface-immobilized single molecule FRET (Förster resonance energy transfer) data follows systematic steps (e.g. single molecule localization, clearance of different sources of noise, selection of the conformational and kinetic model, etc.) that require a solid knowledge in optics, photophysics, signal processing and statistics. The present proceeding aims at standardizing and facilitating procedures for single molecule detection by guiding the reader through an optimization protocol for a particular experimental data set. Relevant features were determined from single molecule movies (SMM) imaging Cy3- and Cy5-labeled Sc.ai5γ group II intron molecules synthetically recreated, to test the performances of four different detection algorithms. Up to 120 different parameterizations per method were routinely evaluated to finally establish an optimum detection procedure. The present protocol is adaptable to any movie displaying surface-immobilized molecules, and can be easily reproduced with our home-written software MASH (multifunctional analysis software for heterogeneous data) and script routines (both available in the download section of www.chem.uzh.ch/rna).
Confocal microscopy is a powerful tool for single molecule investigation of fluorescent macromolecules. Besides the
commonly studied features in single molecule detection, the 3D orientation determination of the emission dipole enables
the analysis of different conformational states. These conformational states can be represented as state depending dipole
orientations intrinsic to the fluorescent molecule and/or in relation to the molecular frame. They might be subject to
intramolecular dynamics, which may lead to spectral diffusion, fluorescence intensity and/or lifetime fluctuations and
changes in the orientation of the emission dipole. We demonstrate a detection scheme that allows for simultaneous
determination of the full 3D emission dipole orientation, the fluorescence intensity, the fluorescence lifetime and the
emission spectra of single fluorescent molecules. We evaluate the feasibility of our approach using pyridyl
functionalized perylene bisimide (PBI) as a model system exhibiting conformational changes. Moreover, MC
simulations demonstrate the full potential of our detection scheme to distinguish between intensity fluctuations due to
conformational changes and changes in the out-of-plane orientation or changes in both of them.